Recent studies from my laboratory have shown that it is possible to propagate and clone serially restimulated murine alloreactive T cells. A variety of new information has arisen from these studies including the concept of novel hybrid histocompatibility determinants present on hybrid (C57BL/6 x A/J)F1 ((B6A)F1) spleen cells. These studies were initiated in order to characterize the receptor(s) on alloreactive T cells. Utilizing similar methodology (serial restimulation with soluble antigen in the presence of syngeneic filler cells followed by soft agar cloning) we have been able to isolate soluble antigen reactive T cell clones. The objectives of the research outlined in this grant proposal are directed at the following major points: 1) Establishment and propagation of clones of murine T cells reactive against well-defined soluble antigens; 2) Biochemical characterization of T cell receptors for antigen utilizing well-defined soluble antigens and T cell clones reactive against these antigens; 3) Studies concerning the mechanisms of MHC restriction and antigen presentation utilizing such cloned cells which recognize antigen only when presented by cells sharing subregions of the MHC with the antigen reactive T cell; 4) Isolation and characterization of immunologically active products secreted from such antigen reactive T cell clones. The methodology utilized for the proposed research is divided into two sections. The first area is isolation and characterization of the antigen-reactive T cell clones using techniques which were originally developed in my laboratory. The second section is the biochemical characterization of products of such cloned antigen reactive T cells. These methods utilize radiolabelling and specific immunoprecipitation of T cell products followed by chemical analysis of immunoprecipitates (polyacrylamide gel electrophoresis, isoelectric focusing and two-dimensional gel electrophoresis). If warranted, we would further analyze such products by peptide mapping and microsequencing in collaboration with Dr. David McKean.